Novel methods for enriching and labeling nucleic acids are needed. For example, gene expression analysis techniques often employ isolation and labeling of ribonucleic acid (RNA). Because of the interest in identifying protein-encoding genes and in examining gene expression levels, it is often desirable to purify or enrich the messenger RNA (mRNA). The poly-adenine 3xe2x80x2-terminus (poly-A tail) of mRNA from eukaryotic cells can be used as a handle to bind to poly(dT) oligonucleotides, and this method is widely used to identify, purify and or label eukaryotic mRNA. However, because prokaryotic mRNA generally lacks poly-A tails, there is a need for alternative methods for purifying and labeling mRNA samples which do not rely on the existence of a poly-A tail.
The presently claimed invention provides methods of preparing a nucleic acid sample for analysis.
In a first embodiment, the presently claimed invention provides a method of preparing a nucleic acid sample for analysis comprising enriching for a population of interest within a mixed population of nucleic acids by contacting the nucleic acid sample with a bait molecule. The bait molecule is capable of complexing specifically to unwanted target sequences within the nucleic acid sample, but is incapable of complexing with sequences from the population of interest. The bait molecule is contacted with the target sequences forming bait:target complexes which are then specifically removed from the nucleic acid sample. The remaining enriched population of interest is then fragmented and a signal moiety is attached to the fragments.
In a second embodiment, the presently claimed invention provides a method of enriching for a population of interest within a mixed population of nucleic acids by contacting the nucleic acid with a bait molecule. The bait molecule is capable of complexing specifically to unwanted target sequences within the nucleic acid sample, but is incapable of complexing with sequences from the population of interest. The bait molecule is contacted with the target sequences forming bait:target complexes which are then specifically removed from the nucleic acid sample. Thus enriching for the population of interest.
In a third embodiment, the presently claimed invention provides a compound having the formula:
n-S-acetyl-PEO-sig 
where n is a polynucleotide, S is a thiol group, acetyl is an acetyl functional group, PEO is polyethelene oxide, and sig is a signal moiety.
In a fourth embodiment, the presently claimed invention provides a method for labeling a polynucleotide comprising contacting the polynucleotide with a PEO-iodoacetyl conjugated to a signal moiety under conditions such that the PEO-iodoacetyl will attach to said nucleotide.
In a fifth embodiment, the presently claimed invention provides a method for labeling a polynucleotide comprising: contacting the polynucleotide with a reactive thiol group to form a thiolated polynucleotide and contacting the thiolated polynucleotide with either a signal moiety capable of reacting with said thiolated polynucleotide under appropriate conditions such that said signal moiety is attached to said polynucleotide.
In a sixth embodiment, the presently claimed invention provides a method for labeling prokaryotic mRNA comprising: obtaining a population of RNA from a prokaryotic organism; enriching the population for mRNA by exposing the population to a plurality of DNA bait molecules which are complementary to at least a portion of the stable RNA in said population under such conditions as to allow for the formation of DNA:RNA hybrids; exposing the DNA:RNA hybrids to RNAse H to remove the RNA from said DNA:RNA hybrids; exposing the remaining DNA to DNase I to remove the DNA, thus producing an enriched population of mRNA; fragmenting the enriched mRNA to form mRNA fragments; exposing the mRNA fragments to (-S-ATP and T4 kinase to produce reactive thiol groups at the 5xe2x80x2 ends of the mRNA fragments; and exposing the thiolated mRNA fragments to PEO-Iodoacetyl-Biotin such that a stable thio-ether bond is formed between said thiolated mRNA fragments and said PEO-Iodoactyl-Biotin.